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Differential Expression of the Human CD8β Splice Variants and Regulation of the M-2 Isoform by Ubiquitination¹

Deepshi Thakral^*, Jessica Dobbins^*, Lesley Devine and Paula B. Kavathas^2,*

^* Department of Laboratory Medicine, Section of Immunobiology, and Department of Genetics and Department of Laboratory Medicine and Immune Monitoring Core Facility, Yale Cancer Center, Yale University School of Medicine, Yale University, New Haven, CT 06520

The CD8β heterodimer functions as a coreceptor with the TCR, influencing the outcome of CD8⁺ T cell responses to pathogen-infected and tumor cells. In contrast to the murine CD8B gene, the human gene encodes alternatively spliced variants with different cytoplasmic tails (M-1, M-2, M-3, and M-4). At present, little is known about the expression patterns and functional significance of such variants. We used quantitative RT-PCR to demonstrate differential mRNA expression patterns of these splice variants in thymocytes and in resting, memory, and activated primary human CD8⁺ T cells. In total CD8⁺ T cells, mRNA levels of the M-1 variant were the most predominant and levels of M-3 were the least detected. The M-4 isoform was predominant in effector memory CD8⁺ T cells. Upon stimulation of CD8⁺ T cells, the M-2 variant mRNA levels were elevated 10–20-fold relative to resting cells in contrast to the other isoforms. Curiously, the M-2 isoform was not expressed on the cell surface in transfected cell lines. Using fluorescent chimeras of the extracellular domain of mouse CD8β fused to the cytoplasmic tails of each isoform, the M-2 isoform was localized in a lysosomal compartment regulated by ubiquitination of a lysine residue (K215) in its cytoplasmic tail. In contrast, upon short-term stimulation, the M-2 protein localized to the cell surface with the TCR complex. The relatively recent evolution of CD8B gene splice variants in the chimpanzee/human lineage is most likely important for fine-tuning the CD8⁺ T cell responses.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work is supported by National Institutes of Health Grant RO1-CA48115 (to P.B.K.) and a Trudeau postdoctoral research fellowship (to D.T.).

² Address correspondence and reprint requests to Dr. Paula B. Kavathas, Yale University School of Medicine, Section of Immunobiology, 300 Cedar Street, The Anlyan Center S641A, P.O. Box 208011, New Haven, CT 06520. E-mail address: paula.kavathas{at}yale.edu

³ Abbreviations used in this paper: MHC-I, MHC class I; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; HA-Ub, hemagglutinin-ubiquitin; LAMP-1, lysosomal-associated membrane protein-1; MFI, mean fluorescence intensity; pMHC-I, peptide-MHC class I; RT, reverse transcriptase; T_EMRA, effector memory RA CD8⁺ T cell.